Among the drugs and agonists that are known to relax the vascular smooth muscle and reduce high blood pressure, beta-adrenergic agonists and nitric oxide-containing compounds are some of the most effective ones. The mechanisms of these drugs are thought to inhibit intracellular free calcium concentration by elevating intracellular cAMP and cGMP, and by activating protein kinases, respectively. However, the molecular targets of cyclic nucleotide-dependent protein kinases are not clear. The molecular basis for the regulation of the vascular smooth muscle by some popular drugs is not well defined. Phospholipase C (PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to generate two important second messengers, diacylglycerol and inositol 1,4,5-triphosphate (InsP3), leading to the activation of protein kinase C and the mobilization of intracellular Ca2+. Detailed characterization of the PLC-B subfamily of proteins will reveal specific receptors, G-proteins, and regulators with which PLC interacts in various signaling pathways, in different developmental and physiological contexts. Thus, PLC enzymes will be used as a "biological probe" for the identification, characterization, and functional dissection of a family of molecular components that collaborates to regulate specific cellular responses in the vascular smooth muscle. Our aims during this grant period are to investigate the regulation of PLC mediated Ca2+ signaling both horizontally, by signaling pathways coupled to cAMP/PKA and cGMP/PKG, and vertically, by interacting with other signaling molecules in the cell. Specifically, we will: 1) Determine the phosphorylation of PLC-B isozymes by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG), respectively. 2) Define the role of protein phosphorylation in G-protein mediated PLC activation and Ca2+ signaling in the vascular smooth muscle cells. 3) Characterize a novel human PLC-B3 interacting protein (hPIP1) in PLC-Ca2+ signaling. Together, we will understand the molecular basis for the modulation of G-protein mediated PLC-Ca2+ signaling in the vascular smooth muscle cells and the regulation of Ca2+ homeostasis in general.